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Bite-Sized Biochemistry #7: Protein Purification & Characterization

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  • Description: Lecture by Kevin Ahern of Oregon State University discussing Biochemistry Basics in BB 450.

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    This course can be taken for credit (wherever you live) via OSU's ecampus. For details, see

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    Related courses include
    BB 350 -
    BB 451 -
    BB 100 -

    Highlights

    1. Nucleic acids (which are negatively charged and 'rod-like' in shape) can be separated by agarose gel electrophoresis readily. In this technique, a 'gel' is made that consists of a matrix material (agarose) that forms a sort of 'mesh' of hole through which the DNA molecules pass. Electric fields are used to separate macromolecules by size. The sample is loaded on the top of the gel and electrical current is passed through it such that the bottom electrode is positive and the top one is negative. Negatively charged DNA molecules at the top of the gel are driven away from the top towards the bottom. Small molecules make their way through the gel fastest and big molecules travel more slowly. The key to gel electrophoresis is to have all of the molecules being separated have a negative charge.

    2. Proteins, however are usually globular in their native state and, without other modification, may be negatively, positively OR neutrally charged. Another type of electrophoresis employs polymers of acrylamide (called polyacrylamide) to form the mesh of the gel. This mesh has smaller holes that agarose and allows separation of smaller molecules like proteins. To separate proteins on the basis of size, the detergent sodium dodecyl sulfate (SDS) is add to the protein mixture, causing the proteins to denature, assume rod-like shapes, and be coated with the negative charge of the SDS. Consequently all proteins in the mixture obtain a negative charge and can be separated just like DNA on SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

    3. Isoelectric focusing is a technique that separates molecules on the basis of their pI (pH at which their net charge is zero). It is performed in tubes containing special compounds (polyelectrolytes) that migrate to specific points in the tube when in the presence of an electric field. This effectively creates a pH gradient from one end of the tube to the other. If proteins are added to the tube as the gradient is getting established, they will migrate to the point in the tube where the pH corresponds to their pI and they will migrate nor further, since they will have a charge of zero.

    4. 2D gel electrophoresis is a powerful tool for proteomics (study of all the proteins of a cell) that combines the techniques of isoelectric focusing with SDS-PAGE. In this method, proteins are first separated according to their pI by isoelectric focusing. Then the tube from the isoelectric focusing is mixed with SDS and applied to the top of an SDS-PAGE gel where the proteins are separated by size. The result is a two dimensional separation of virtually every protein in the cell. These protein 'spots' can be cut out of the gel, the protein can be extracted and then it can be characterized by MALDI-TOF (covered in next lecture) to identify it.

    5. During purification of proteins it is important to follow the purification process. At each step of the purification, a small sample of the protein extract is taken and the total amount of protein, and the amount of activity of the desired protein are measured. The specific activity of the protein in the tube is the amount of activity (in units) divided by the total mass of protein. The yield of the desired protein at any point in the purification process is the number of units of the desired protein at that point dividied by the number of units that one started with. The purification level at any point is the specific activity at that point divided by the specific activity one started with.

    6. Amino acids in proteins can be released by breaking the peptide bonds between them using high temperature and an acid. When this happens, one can use HPLC to measure the amount of each amino acid in the protein.

    7. Breaking large proteins down into smaller pieces is also important for studying them. Some reagents include cyanogen bromide (breaks bonds on carboxyl side of methionine residues in a protein) and enzymes known as proteases. One such protease is trypsin, which cleaves on the carboxyl side of lysine and arginine residues in a polypeptide. Carboxypeptidase is another enzyme that breaks peptide bonds. It cleaves them on the amino side of the carboxy-terminal amino acids.
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